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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Short description of key information:
In vitro:
Two Ames tests are available: In one test 5 strains were tested, in the other 4 strains were used.
Additionally, the test item was tested in an HPRT assay according OECD Guideline study 476. Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and therefore was considered to be non-mutagenic in this HPRT assay (Hall/ Harlan/ Lanxess, 2010).

In vivo:
In a micronucleus test according to OECD guideline 474 no indications of a clastogenic effect of the test substance dichlorotoluene (mixture of isomers) were found after a single intraperitoneal treatment with 1500 mg/kg (Herbold/ Bayer AG, 1993).

Overall, it can be concluded that the compound is not genotoxic in vitro or in vivo.

Endpoint Conclusion: No adverse effect observed (negative).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The test item 'Dichlortoluol Gemisch' was initially investigated for point mutagenic effects in the plate incorporation test. The test item, dissolved in DMSO, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471.
The test item was investigated in an independent repeat using the preincubation modification in doses of up to and including 500 µg per plate without and with S9 mix. Other experimental conditions remained unchanged.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA102
Additional strain / cell type characteristics:
other: All S. typhimurium strains were checked for of rfa-mutation ("deep rough"), for their resistance to ampicillin to establish the presence ofthe R-factor/pKM101-plasmid and for their resistance to tetracycline to establish the presence ofthe pAQ1-plasmid
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
plate incorporation test: up to and including 5000 µg per plate without and with S9 mix
preincubation method: up to and including 500 µg per plate without and with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-Nitro-1,2-phenylene diamine, 2-aminoanthracene
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Doses up to and including 160 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: TA1535, TA100, TA1537, TA98 and TA102

Evidence of mutagenic activity ofthe test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

Conclusions:
Interpretation of results: negative
Executive summary:

The test item 'Dichlortoluol Gemisch' was initially investigated for point mutagenic effects in the plate incorporation test. The test item, dissolved in DMSO, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471. Doses up to and including 160 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used strain-specific up to 5000 µg per plate for assessment purposes.

The test item was investigated in an independent repeat using the preincubation modification in doses of up to and including 500 µg per plate without and with S9 mix. Other experimental conditions remained unchanged. Doses up to and including 16 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used strain-specific up to 500 µg per plate for assessment purposes.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

Therefore, the test item was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study , only 4 strains tested
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Only 4 strains tested.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames Test
Species / strain / cell type:
other: S. typhimurium TA 98, 100, 1535, 1537
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
up to 5000 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide for TA 1535, Nitrofurantoin for TA 100, 4-nitro-1,2-phenylene diamine for TA 1537 and TA 98, 2-aminoanthracene serves as a control for the activating effect of the S9 mix.
Details on test system and experimental conditions:
Ames test
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Summary of the Results With Dichlorotoluene (Isomer mixture) in the Salmonella/Microsome Test:

 S9 mix  TA 1535  TA 100  TA 1537  TA 98
 without  negative  negative  negative  negative
 with  negative  negative  negative  negative
Conclusions:
Interpretation of results: negative
Executive summary:

The test substance was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four histidine-auxotrophic Salmonella typhimurium LT2 mutant strains (TA 1535, TA 100, TA 98, and TA 1537).

Doses up to and including 50 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong bacteriotoxic effect, but this range could nevertheless be used for assessment purposes.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.
GLP compliance:
yes
Type of assay:
other: GENE MUTATION ASSAY IN CHINESE HAMSTER V79 CELLS IN VITRO
Target gene:
HPRT gen-lokus
Species / strain / cell type:
other: CHINESE HAMSTER V79 CELLS
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment I (concentrations in µg/mL)
Exposure period: 4 hours, without S9 mix, 3.1, 6.3, 12.5, 25.0, 37.5, 50.0, 75.0;
Exposure period: 4 hours, with S9 mix, 12.5, 25.0, 50.0, 100.0, 150.0, 200.0, 300.0;
Experiment II (concentrations in µg/mL)
Exposure period: 24 hours, without S9 mix, 5.0, 10.0, 20.0*, 40.0, 60.0 ,80.0, 100.0*;
Exposure period: 4 hours**, with S9 mix, 50.0, 100.0, 150.0, 175.0, 200.0 250.0, 300.0;
Exposure period: 4 hours, with S9 mix, 9.4 18.8, 37.5, 75.0, 150.0, 200.0, 250.0

* was evaluated for mutagenicity only in one culture
** was repeated due to strong cytotoxicity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: EMS; ethylmethane sulfonate , With metabolic activation: DMBA; 7,12-dimethylbenz(a)anthracene
Species / strain:
other: CHINESE HAMSTER V79 CELLS
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The test item Dichlortoluol-Gemisch was assessed for its potential to induce gene muta-tions at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental conditions. In the first experiment the treatment period was 4 hours with and without meta-bolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the test item applied in the pre-experiments equals a molar concentration of 10 mM, with respect to the purity of the test item (98.4 %). Due to strong test item induced cytotoxicity the concentrations applied in the main experiments were lower. In the main experiments no precipitation of the test item was observed up to the maximum concentration in all experiments. The maximum concentrations evaluated for mutagenicity were limited by strong cytotoxic effects. Except of the experimental part with S9 mix in experiment II the survival and/or the relative cell density were reduced to below 20 % in the highest evaluated dose group.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. Predominantly the mutant fre-quencies remained well within the historical range of solvent controls. In experiment I in the absence of S9 mix and in experiment II in culture II several values (32.7 to 66.3 mutant colonies/106 cells) exceeded the respective solvent control data ranges. These values were either not reproducible in the parallel culture or were only slightly higher than the corresponding historical data range (experiment I, treatment with 6.3 µg/mL in the absence of S9 mix). The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of the experiment I without metabolic activation at 6.3 µg/mL and in the first culture of experiment II at 10.0 and 20.0 µg/mL (without S9 mix). This effect however, was judged to be based upon the rather low solvent controls of 9.7 and 7.6 mutant colonies/106 cells, respectively.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture of the first experiment without metabolic activation. This trend however, was judged as irrelevant since it actually was reciprocal, going down versus increasing concentrations.

In both experiments of this study (with and without S9 mix) the range of the solvent con-trols was from 7.6 up to 39.0 mutants per 106 cells; the range of the groups treated with the test item was from 0.0 up to 66.3 mutants per 106 cells.

The mutagenicity frequencies of the solvent controls in experiment I, culture I and experi-ment II, culture II without metabolic activation (39.0 and 32.1 mutant colonies per 106 cells) slightly exceeded the historical range of solvent control (0.6 to 32.4 and 2.2 – 31.5 mutant colonies per 106 cells). However, this effect was judged as irrelevant since it is very minor and the corresponding solvent control in the parallel cultures remained well within the range of historical controls.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the ex¬perimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Dichlortoluol-Gemisch is considered to be non-mutagenic in this HPRT assay.

Conclusions:
Interpretation of results: negative
Executive summary:

The study was performed to investigate the potential of Dichlortoluol-Gemisch to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activa-tion and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration in the pre-experiment (1640.0 µg/mL) was equal to a molar concentration of about 10 mM, with respect to the purity of the test item (98.4 %). The dose range of the main experiments was limited by test item induced cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Dichlortoluol-Gemisch is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Short description of key information:
In vivo:
In a micronucleus test according to OECD guideline 474 no indications of a clastogenic effect of the test substance dichlorotoluene (mixture of isomers) were found after a single intraperitoneal treatment with 1500 mg/kg (Herbold/ Bayer AG, 1993).

Endpoint Conclusion: No adverse effect observed (negative).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Route of administration:
intraperitoneal
Duration of treatment / exposure:
single treatment
Frequency of treatment:
single treatment
Post exposure period:
Time of sacrifice:
negative control: 24 hrs,
test substance: 16, 24, 48 hrs,
positive control cyclophosphamide: 24 hrs
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid

Summary of results of Micronucleus test with Dichlorotoluene (mixture of isomers) (after acute intraperitoneal treatment with 1500 mg/kg body weight:

          micronucleated cells per 1000
 experimental groups  number of evaluated polychromatic erythrocytes  number of normochromatic erythrocytes per 1000 polychromatic erythrocytes  normochromatic erythrocytes  polychromatic erythrocytes
 negative control  10.000  1138 + 460  2.0 + 2.5  2.0 + 2.2
 test substance 16 hrs  10.000  977 + 232  1.9 + 1.3  1.7 + 1.5
 test substance 24 hrs  10.000  1149 + 243  1.9 + 1.2  2.0 + 1.8
 test substance 48 hrs  10.000  870 + 242  2.2 + 2.0  2.3 + 1.8
 positive control CP 20 mg/kg 10.000   728 + 227  1.6 + 1.4  19.1* + 10.4

*P< 0.01 in non-parametric Wilcoxon ranking test

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

This micronucleus test was conducted to investigate the test substance dichlorotoluene (mixture of isomers) in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as control.

The treated animals received a single intraperitoneal administration of either the test substance or cyclophosphamide. The femoral marrow of groups treated with the test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of the test substance and the positive control, cyclphosphamide, were 1500 and 20 mg/kg body weight, respectively.

The animals treated with the test substance showed symptoms of toxicity (apathy, stretching of body, roughed fur, staggering gait, spasm and difficulty in breathing) after administration. However, all animals survived untill the end of the test.

There was no altered ratio between polychromatic and normochromatic erythrocytes.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

No indications of a clastogenic effect of the test substance dichlorotoluene (mixture of isomers) were found after a single intraperitoneal treatment with 1500 mg/kg.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro:

Ames Test by Nern, 2014: The test item 'Dichlortoluol Gemisch' was initially investigated for point mutagenic effects in the plate incorporation test. The test item, dissolved in DMSO, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471. Doses up to and including 160 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used strain-specific up to 5000 µg per plate for assessment purposes.

The test item was investigated in an independent repeat using the preincubation modification in doses of up to and including 500 µg per plate without and with S9 mix. Other experimental conditions remained unchanged. Doses up to and including 16 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used strain-specific up to 500 µg per plate for assessment purposes.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied. Therefore, the test substance was considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test (Nern/Bayer AG, 2014).

Ames test by Gahlmann, 1992: The test substance was investigated using the Salmonella/microsome test according to OECD guideline 471 for point mutagenic effects in doses up to 5000 µg per plate on four histidine-auxotrophic Salmonella typhimurium LT2 mutant strains (TA 1535, TA 100, TA 98, and TA 1537).

Doses up to and including 50 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong bacteriotoxic effect, but this range could nevertheless be used for assessment purposes.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic with and without S9 mix in the Salmonella/microsome test (Gahlmann/Bayer AG, 1992).

The study was performed to investigate the potential of Dichlortoluol-Gemisch to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration in the pre-experiment (1640.0 µg/mL) was equal to a molar concentration of about 10 mM, with respect to the purity of the test item (98.4 %). The dose range of the main experiments was limited by test item induced cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Dichlortoluol-Gemisch is considered to be non-mutagenic in this HPRT assay (Hall/ Lanxess, 2010).

In vivo:

A micronucleus test according to OECD guideline 474 was employed to investigate the test substance dichlorotoluene (mixture of isomers) in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as control.

The treated animals received a single intraperitoneal administration of either the test substance or cyclophosphamide. The femoral marrow of groups treated with the test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of the test substance and the positive control, cyclphosphamide, were 1500 and 20 mg/kg body weight, respectively.

The animals treated with the test substance showed symptoms of toxicity (apathy, stretching of body, roughed fur, staggering gait, spasm and difficulty in breathing) after administration. However, all animals survived until the end of the test.

There was no altered ratio between polychromatic and normochromatic erythrocytes.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

No indications of a clastogenic effect of the test substance dichlorotoluene (mixture of isomers) were found after a single intraperitoneal treatment with 1500 mg/kg (Herbold/ Bayer AG, 1993).


Short description of key information:
In vitro:
Two Ames tests are available: In one test 5 strains were tested, in the other 4 strains were used.
Additionally, the test item was tested in an HPRT assay according OECD Guideline study 476. Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and therefore was considered to be non-mutagenic in this HPRT assay (Hall/ Harlan/ Lanxess, 2010).

In vivo:
In a micronucleus test according to OECD guideline 474 no indications of a clastogenic effect of the test substance dichlorotoluene (mixture of isomers) were found after a single intraperitoneal treatment with 1500 mg/kg (Herbold/ Bayer AG, 1993).

Overall, it can be concluded that the compound is not genotoxic in vitro or in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All available in vitro and in vitro tests are negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification for genetic toxicity is not justified.